Intramolecular and Intermolecular Interactions of Protein Kinase B Define Its Activation In Vivo

Abstract

Protein kinase B (PKB/Akt) is a pivotal regulator of diverse metabolic, phenotypic, and antiapoptotic cellular controls and has been shown to be a key player in cancer progression. Here, using fluorescent reporters, we shown in cells that, contrary to in vitro analyses, 3-phosphoinositide–dependent protein kinase 1 (PDK1) is complexed to its substrate, PKB. The use of Förster resonance energy transfer detected by both frequency domain and two-photon time domain fluorescence lifetime imaging microscopy has lead to novel in vivo findings. The preactivation complex of PKB and PDK1 is maintained in an inactive state through a PKB intramolecular interaction between its pleckstrin homology (PH) and kinase domains, in a “PH-in” conformer. This domain–domain interaction prevents the PKB activation loop from being phosphorylated by PDK1. The interactive regions for this intramolecular PKB interaction were predicted through molecular modeling and tested through mutagenesis, supporting the derived model. Physiologically, agonist-induced phosphorylation of PKB by PDK1 occurs coincident to plasma membrane recruitment, and we further shown here that this process is associated with a conformational change in PKB at the membrane, producing a “PH-out” conformer and enabling PDK1 access the activation loop. The active, phosphorylated, “PH-out” conformer can dissociate from the membrane and retain this conformation to phosphorylate substrates distal to the membrane. These in vivo studies provide a new model for the mechanism of activation of PKB. This study takes a crucial widely studied regulator (physiology and pathology) and addresses the fundamental question of the dynamic in vivo behaviour of PKB with a detailed molecular mechanism. This has important implications not only in extending our understanding of this oncogenic protein kinase but also in opening up distinct opportunities for therapeutic intervention. Regulation of intracellular signaling depends on the precise operation of molecular switches such as kinases and phosphatases. Disruption of their activities leads to inappropriate cellular proliferation, growth, and survival. Protein kinase B (PKB) is a critical kinase that regulates events downstream of growth factor receptors. It is also involved in human cancer, where its overexpression induces malignant transformation. We studied the molecular mechanisms of PKB's interaction with its upstream regulator, 3-phosphoinositide–dependent protein kinase 1 (PDK1). By using a fluorescent probe, we monitored the conformational changes of PKB in cells using Förster resonance energy transfer detected by fluorescence lifetime imaging microscopy. Applying this approach, we show that PKB and PDK1 are found as complexes in the cytoplasm. Despite this proximity to its regulator, we show that PKB remains inactive through an intramolecular interaction of its pleckstrin homology (PH) domain and kinase domain. We refer to this inactive state as the “PH-in” conformer. Following growth factor activation, PKB changes conformation to the “PH-out” conformer and is phosphorylated by PDK1. The active “PH-out” conformer dissociates from the plasma membrane to phosphorylate downstream proteins. Our in vivo studies provide a new model for the mechanism of PKB.